IL125314A - Processes for coupling amino acids using bis-(trichloromethyl) carbonate - Google Patents

Abstract

A process of coupling an amino acid residue to a peptide chain which comprises: providing an amino acid residue having a free carboxylic group and blocked amino group, optionally having additional blocked functional side chains; reacting the blocked amino acid with bis- (trichloromethyl) carbonate in a solvent inert to this reaction to obtain an amino acid chloride; neutralizing the free acid with an organic base; adding the resulting suspension containing the amino acid chloride to a compound selected from the group consisting of a peptide having a blocked carboxyl terminus and a free amino terminus, and a peptidyl resin having at least one free amino terminus; and providing reaction conditions enabling the coupling of the amino acid chloride to the peptide to yield a peptide elongated by one amino acid residue, or providing reaction conditions enabling the coupling of the amino acid chloride to the solid support.

Description

mp p'nnnftTiBj-o'a niusnNa ΙΓΠΝ nianin mnsirt D'D nn PROCESSES FOR COUPLING AMINO ACIDS USING BIS-(TRICHLOROMETHYL) CARBONATE P/010 :WJ1 PROCESSES FOR COUPLING AMINO ACIDS USING BIS-(TRICHLOROMETHYL) CARBONATE FIELD OF THE INVENTION The invention relates to a process for the in situ generation of amino acid chlorides utilizing bis- (trichloromethyl) carbonate, commonly known as triphosgene, and to methods of using this process for solid phase peptide synthesis and for derivatization of a solid support.

BACKGROUND OF THE INVENTION In the field of peptide synthesis certain couplings are known as difficult couplings, especially those involving coupling to bulky or sterically hindered amino acid residues, such as N alkylated, C-alkylated and C branched amino acids. In order to obtain acceptable yields when these couplings are performed a variety of special coupling reagents have been developed. Among other known procedures, is the use of pre-formed amino acid chlorides to improve the outcome of the coupling reactions.

The general use of protected amino acid chlorides in solid phase peptide synthesis (SPPS) is limited mainly because of the fact that chlorides of fluorenylmethoxycarbonyl Fmoc) amino acid having side chains protected with acid labile protecting groups, including but not limited to t-butyl (t-Bu), t-butoxycarbonyl (Boc) or trityl (Trt), have limited shelf stability. For example, chlorides of Fmoc-amino acids (AAs) with t-Bu-protected side chains could not generally be accommodated. In some cases (aspartic acid and glutamic acid) the chlorides could not be obtained and in other cases (tyrosine, serine, threonine) their shelf stability appeared insufficient for practical utilization. In addition, the preparation of chlorides derived from Fmoc- Lysine(Boc), Fmoc-Tryptophan (Boc), Fmoc-Cysteine(Trt), Fmoc-Glutamine(Trt) and Fmoc-Arginine 2,2,5,7,8-Pentamethyl chroman-6-sulphonyl (Pmc) is problematic because of side reactions and require special reaction conditions and purification (Carpino et al., 1996). This problem also hampers the general use of pre-formed Fmoc amino acid chlorides in automatic peptide synthesis. Despite these limitations, acid chlorides were used in SPPS especially for the assembly of hindered secondary amino acids (see Carpino et al., 1996 and ref. within) Coupling of protected amino acids to Na -alkylated amino acids was previously considered to be a difficult coupling both in solution and solid phase. This coupling was used in model peptides to demonstrate the efficiency of new, more effective, coupling methods. In these models, N-Me amino acids were used as nucleophiles, since coupling to N-Me amino acids having steric hindrance on the C (e. g. N-methyl valine and N-methyl Aminoisobutyric acid) was found to be much slower than to proline. Certain coupling agents and activation * methods such as bromo-tris-pyrrolidino-phosphonium hexafluorophosphate ( PyBroP) (Coste et al., 1990), l-hydroxy-7-azabenzotriazole (HOAt)/O-(7-azabenzotriazol-l-yl)-l, 1,3,3-tetramethyluronium hexafluorophosphate (HATU) (Carpino et al., 1994),urethane- protected N-carboxyanhydrides (UNCA) (Spencer et al., 1992) and acid halides (Carpino et al., 1996) were specially recommended to achieve coupling to N-alkyl amino acids.

The acid chloride method was found to be a superior way to couple protected amino acids to sterically hindered amino acid derivatives, such as the N-alkyl amino acids during SPPS of backbone cyclic peptides. To overcome the limitations of the pre-formed acid chloride method and to allow its general use in SPPS, it would be advantageous to have an efficient and generally applicable method allowing the in-situ generation of Fmoc-AAs chlorides.

The reagent bis-(trichloromethyl)carbonate (BTC) (Councler, 1880) also named hexachlorodimethyl carbonate or "triphosgene" is a solid stable phosgene substitute equivalent to three moles of phosgene.

Triphosgene has been used as an efficient carbonylating agent for liquid and solid phase synthesis of various aza-analogues of peptides containing aza-alanine, aza-aspartic acid and aza-asparagine residues (Andre et al., 1997).

The use of triphosgene as a reagent for formation of isocyanates or other reactive species useful in peptide chemistry has also been disclosed (Eckert DE3440141, Nippon Kayaku JP 10007623). The usefulness of triphosgene in preparation of various intermediates for pharmaceuticals has also been disclosed (Hoffmann et al. DD292452).

It is neither taught nor suggested in the art that the triphosgene reagent is suitable for the in-situ generation of protected amino acid chlorides, namely as a coupling agent in SPPS (for review see Cotarca et al., 1996) Phosgene gas has long been a valuable asset to both lab and plant scale operations however the dangers of using it are also well documented, especially the respiratory hazards. Liquid trichloromethyl chloroformate, commonly known as "diphosgene" (Fridgen and Prol, 1989) which has already been used as a phosgene substitute, has proven useful in all common phosgene reactions, but being a liquid its transport and storage still impose considerable hazard. Being a crystalline solid (mp 81-83 ^C), BTC is safer and easy to handle and therefore became the reagent of choice for all applications where phosgene chemistry is required (Cotarca et al., 1996). Synthetically, one mole of BTC yields three mole-equivalents of phosgene which reacts with hydroxyl, amine or carboxylic acid nucleophiles forming chloroformate, isocyanate or acyl chloride, respectively. Considering all these features together with the fact that BTC is inexpensive and less susceptible to hydrolysis than phosgene, it is surprising that the use of BTC as a general coupling agent has not been considered.

Backbone cyclized peptide analogs Backbone cyclization (BBC) is a concept that allows the conversion of peptides into conformationally constrained peptidomimetics with desired pharmacological properties such as metabolic stability, selectivity and improved bioavailability (Gilon et al., 1991; Byk et al., 1996; Gilon et al., 1998).

In backbone cyclization the Na and/or Ca atoms in the peptide backbone are linked through various spacers. To synthesize N-backbone cyclic (N-BBC) peptides a large number of orthogonally protected -functionalized Na alkyl amino acids (N-BBC building units, BU) were prepared (Bitan et al. 1997a; Muller et al. 1997). These units were incorporated into peptides by SPPS or solution methodologies and after orthogonal removal of the protecting groups from the ω-functions on the Na -alkyl they are cyclized on the resin. A critical step in the synthesis of N-BBC peptides is the coupling of protected amino acids to the sterically hindered secondary amine of the Να (ω -functionalized alkyl) amino acid residue on the peptidyl-resin.

The synthesis of N-BBC peptides that incorporate N (ω-functionalized alkyl) Glycine building units were reported previously. In these cases couplings of the protected amino acids to the secondary amine of the Gly building unit attached to peptidyl resin were achieved by multiple couplings with benzotriazole-l-yl-oxy-tris-(dimethylamino)-phosphonium hexaflourophosphate BOP or PyBroP as coupling agents (Bitan et al., 1997b; Byk et al., 1996) Generally, the coupling of protected AAs to building units other than Gly (non-Gly BBC building units) were found to be difficult and even impossible. Consequently, the formation of a reduced peptide bond to the building unit was advocated (Kaljust and Unden, 1994).

We have shown that the coupling of many Fmoc AAs to a variety of non-Gly building units attached to peptidyl-resin could be achieved in moderate to high yields using the acid-chloride method but not acid fluorides (Carpino et al., 1996) or other coupling agents such as PyBrOP (Coste et al., 1990), HOAt/HATU (Carpino, 1993; Carpino et al., 1994), 2-(2-Oxo-l(2H)-pyridyl)-l, 1,3,3-bispenta-methyleneuronium tetrafluoroborate (TOPPipU) (Henklein et al., 1990), UNCA (Spencer et al., 1992) and Mukaiyama reagent (Mukaiyama, 1979).

SUMMARY OF THE INVENTION It is an object of the present invention to provide a process for the improvement of difficult couplings in SPPS. It is a further object of this invention to provide a method for improving the yield of the desired stereoisomer in solid phase peptide synthesis. It is yet a further object to provide methods for facilitating multiple parallel peptide synthesis ( PPS).

It is yet a further object to provide methods for attaching protected amino acids to functionalized solid supports or to attach biomedically important ligands to a peptide or peptidyl resin during SPPS. It is yet a further object to provide methods to cyclize peptides attached to a solid support.

According to the present invention a process is provided for the in situ generation of protected amino acid chlorides, by use of an agent such as phosgene, diphosgene or more preferably triphogene. The protected amino acid chlorides thus generated are particularly useful in the coupling of an amino acid residue to a peptide chain. They can also be used for the coupling of a carbohydrate moiety to a peptide chain.

The in situ generation of acid chlorides using the methods of the present invention thus further provides a process whereby other biologically important acids, including but not limited to glucoronic acid, DTP A, and DOTA, may be connected to a peptide chain through the amine backbone or through an amino acid side chain functionality.

It has now been found that in accordance with the principles of the present invention bis-(trichloromethyl)carbonate, also known by the trivial chemical name triphosgene, can be used for the in situ generation of protected amino acid chlorides. The use of this process overcomes many of the problems encountered in difficult coupling steps in SPPS, particularly where the coupling step involves sterically hindered amino acid residues or bulky amino acid analogs.

Methods are provided for the use of BTC as a convenient and efficient coupling agent for difficult couplings in SPPS. These methods provide greatly enhanced yields of the desired product in SPPS with retention of configuration and without undesired side reactions and also facilitate the performance of multiple parallel peptide synthesis.

One method according to the present invention provides a process of coupling an amino acid residue to a peptide chain comprising: providing an amino acid residue having a free carboxylic group and blocked amino group, optionally having additional blocked functional side chains; reacting the blocked amino acid with of bis-(trichloromethyl)carbonate in an solvent inert to this reaction to obtain an amino acid chloride; neutralizing the free acid by addition of an organic base; adding the resulting suspension containing the amino acid chloride to a compound selected from the group consisting of a peptide having a blocked carboxyl terminus and a free amino terminus, and a peptidyT resin having at least one free amino terminus; providing reaction conditions enabling the coupling of the amino acid chloride to the peptide to yield a peptide elongated by one amino acid residue.

A second method according to the present invention provides a process of coupling an amino acid residue to a solid support comprising: providing an amino acid residue having a free carboxylic group and blocked amino group, optionally having additional blocked functional side chains; reacting the blocked amino acid with bis-(trichloromethyl)carbonate in a solvent inert to the reaction to obtain an amino acid chloride; Neutralizing the free acid by addition of an organic base; adding the resulting suspension containing the amino acid chloride to a compound selected from the group consisting of a resin having at least one free amino terminus and a solid support having a functional group capable of binding the chloride; providing reaction conditions enabling the coupling of the amino acid chloride to the solid support.

A currently most preferred embodiment according to the present invention is summarized in the following scheme: Scheme: Difficult coupling using BTC in solvent inert to this reaction Fmoc-AA-OH + BTC I Π .

R Fmoc-AA-N-CH-CO-Peptidyl -Resin R BTC = Bis-(trichloromethyl)carbonate (triphosgene) Fmoc-AA = Fluorenylmethoxycarbonyl-proteinogenic-a-Amino Acid R = Side chains of all proteinogenic a- amino acids R' - CH3, (CH2)n=2.4-NH-Alloc, (CH2)n=2.3-COOAllyl R3,rN = tertiary or aromatic amine BRIEF DESCRIPTION OF THE FIGURES Figure 1. HPLC chromatogram of the product obtained in example 38.

Figure 2. Mass spectra analysis of the product obtained in example 38.

Figure 3. HPLC chromatogram of the product obtained in example 49.

Figure 4. HPLC chromatogram of the product obtained in example 55.

DETAILED DESCRIPTION OF THE INVENTION According to the present invention processes for the in situ generation of protected amino acid chlorides are provided utilizing an agent such as phosgene, diphosgene or triphosgene. In all the specific preferred embodiments presented herein the processes are exemplified utilizing solid phase peptide synthesis although these processes are also highly suitable to the synthesis of peptides in solution or for the methods of multiple parallel peptide synthesis as are known in the art. Moreover, the same processes can potentially be used to the coupling of other moieties to a peptide chain, including but not limited to the coupling of a carbohydrate moiety to a peptide chain.

For the majority of examples which follow, certain procedures were used throughout, which have therefore, for the sake of simplicity, been summarized herein under the heading general procedures.

General Procedure A: SPPS of model peptides l g Rink Amide methylbenzhydrilamine (MBHA) resin (0.55 mmol/g) were swelled for 1.5 hour in N-methyl pyrollidone (NMP) in a reaction vessel equipped with sintered glass bottom and placed on a shaker. The Fmoc protection was removed with 20% piperidine in NMP (twice fifteen minutes, 8 mL each). After washing with NMP (5 times, 8mL for 2 minutes), the Fmoc removal was monitored by Kaiser test.

A coupling cycle was carried out with Fmoc AA or Fmoc N-Me AA or Fmoc Building Unit (BU, 3 equivalents), bromo-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBrOP, 3 equivalents) diisopropylethylamine (DIEA) (6 equivalents) in NMP (8 mL) for lhour at room temperature. Reaction completion was monitored by Kaiser test and the solvent was removed by filtration. The resin was washed with NMP (5 times, 8mL 2 minutes). The Fmoc protecting group was removed as above. Coupling of Fmoc AA or Fmoc N-Me AA or Fmoc BU to AA-resin was performed as described above, using PyBrOP as coupling agent. Coupling of Fmoc AA or Fmoc N-Me AA or Fmoc BU to N-Me or BU-resin was performed as described in General procedure B. Peptide elongation was performed by repeating the removal of the Fmoc protecting group and the coupling cycle described above.

* Final Fmoc deprotection was followed by washes (NMP 5 times, 8mL 2 minutes and dichloromethane (DC ) 3 times, 8mL 2 minutes). The peptidyl resin was dried in vacuum. Fast cleavage: a small amount of peptidyl resin was treated with 95% trifluouroacetic acid (TFA) containing 1% triisopropylsilane (TIS). The solvents were removed by a stream of nitrogen and the residue was taken up in water: acetonitrile (ACN containing 0.1%TFA. After filtration the solution was injected to UPLC and/or to the mass-spectrometer.

General Procedure B : Coupling of Fmoc-AA's to AA peptidyl resin and to N-alkylated AA peptidyl resin using BTC Fmoc AA or Fmoc N-Me AA or Fmoc BU (5 eq, 0.275 mmol) and BTC (1.65 eq, 0.09 mmol) were dissolved in either tetrahydrofuran (THF), dioxane, diglyme, or in 1,3-dichloropropane to give 0.15 M solution, to which 2,4,6-collidine (14 eq, 0.75 mmol) was added to give a white suspension. This suspension was poured into the N-Me AA or BU's -peptidyl-resin prewashed with the appropriate solvent. The mixture was shaken at 50° C for 1 hour and filtered. The peptidyl resin was washed with DCM, swelled with the appropriate reaction solvent, and the coupling repeated once more. In cases where coupling was performed with Fmoc-AA's to AA peptidyl resin, only 3 eq of Fmoc-AA's ,1 eq of BTC and 8 eq of 2,4,6-collidine were used in dioxane at room temp for lh.

General Procedure C : Coupling of Fmoc- AA or NMeAA or BU to functionalized solid support Fmoc AA or Fmoc NMeAA or Fmoc BU chloride was prepared in situ as described in Procedure B. The suspension in dioxane was poured onto preactivated glass or onto hydroxymethylated polystyrene. The mixture was shaken at 50° C for 1 hour and filtered. The derivatized support was washed with DCM. The degree of derivatization was determined by the Fmoc piperidine method.

General Procedure D : Derivatization of amino or hydroxy peptidyl resin Acids such as 1,2,3,4-tetra-O-acetyl glucoronic acid, diethylentriaminepenta acetic acid (DTP A), tetraazacyclodecanetetraacetic acid ( DOT A) or parahydroxy phenyl propionic acid (FJPPA) were converted to their corresponding acid chlorides using BTC as described in procedure B. The suspension was poured into amino or hydroxy peptidyl resin and heated lh. at 50° C. After filtration and wash, the derivatized peptidyl resin was cleaved and characterized as described in procedures A, G and H.

General Procedure E: SPPS of Backbone Cyclic Peptides Peptidyl-resins that contain two BUs were synthesized according to General procedures A & B. The Allyl/Allyloxycarbonyl Alloc protecting groups were removed by reaction with Tetrakis/triphenylphosphine)-palladium, acetic acid 5x and N-methylmorpholine 2.5% in DCM under argon, for 1.5 hours at room temperature. The peptide resin was washed with MP as above. Cyclization was carried out in NMP with PyBrOP (3 equivalents) and DIEA (6 equivalents for 1 hour at room temperature. After washings with NMP the cyclization was repeated. The backbone cyclic peptide was deprotected and cleaved from the resin by treatment with 10 mL of TFA 94%, water 2.5%, TIS 1% and ethanedithiol 2.5% at 0 °C under argon for 0.5 hour and at room temperature for 1-3 hours. The resin was removed by filtration and washed with additional amount of TFA, the combined solution evaporated by a nitrogen stream to give an oil which upon treatment with cold ether (40mL) solidified. The ether removed after centrifugation and the solid dried in high vacuum overnight to give the crude peptide.

General Procedure F : Cyclization of peptides containing two N-BU's Peptidyl-resins that contain two N-BUs were synthesized according to General procedures A, B and E. Cyclization was carried out in dioxane with BTC (0.33 eq) at room temp' for lh. Chemical procedures and characterization were done as described in General Procedure E, G and H.

General Procedure G: HPLC analysis of crude peptides A sample of the crude peptides was dissolved in solvent A (water+0.1% TFA) and injected into the HPLC machine (column C18 250X4mm, flow lmL/minute). Eluent solvent A and B (CAN, 0.1% TFA). Hydrophobic peptides were eluted using linear gradient from 90% to 10% A in 35 minutes. Hydrophilic peptides were eluted using linear gradient from 100· to 10% A in 35 minutes. Peptides were detected by an online UV detector set at 214nm.

General Procedure H: Mass spectral analysis of peptides Crude peptides or fractions collected from the HPLC were analyzed by quadrupole or ion trap mass spectrometers.

The invention will be exemplified with regard to particular peptides and peptidomimetic compounds. These examples are to be construed in a non-limitative fashion, and it is understood that the invention is not limited by the scope of the examples, but rather by the scope of the claims which follow the specification.

EXAMPLES Carboxylic acid chlorides are most conveniently prepared when BTC is reacted with carboxylic acids in an solvent inert to BTC at temperatures ranging from slightly above room temperature to reflux in the presence of DMF or tertiary amines as catalysts. With that in mind and the fact that in the case of the pre-prepared Fmoc-AA's chlorides best conversions were obtained by heating in NMP, our preliminary studies with BTC were conducted under the following conditions: Fmoc-AA's (3 eq) and BTC (1.2 eq) were reacted in NMP followed by DIEA (12 eq) addition. After half an hour at rt the activated AA's were poured into the peptide resin which was swelled at 65®C for lh. Double coupling under these conditions gave the desired peptides but unfortunately with the loss of stereochemistry. Changing the base to the weaker sterically hindered 2,4,6-collidine did not improve the coupling stereointegrity much, as shown in Table 1.

Table 1. Summary of difficult couplings using BTC NMP at 65°C In entry 1-2, DIEA was used as base. R.T of substrate is for the Fmoc-protected derivative. Entries 3-10 were performed in MPS format in 5.5 mM scale with collidine as base. a. Based on integration of area under the curve obtained from HPLC analysis. b. Analyzed according to general procedure G for hydrophobic peptides. c. Which have identical mass but different retention times. 125314/2 To explain the racemization of Fmoc AA's during their efficient BTC mediated coupling to BU-peptidyl-resins in NMP we suggest a mechanism (Scheme 1) in which excess NMP reacts with BTC to form 1 which by loosing phosgene and CO2 furnished Vilsmayer-type intermediate 2 //wO-attack by the Fmoc-AA on this chloroiminium ion 2 yields active ester 3, who now couple to the peptide resin. On the other hand, N-alkoxycarbonyl protected amino active ester 3 under base catalysis is known to transform to oxazolone 4 which is undergoes ready racemization.

Further experiments with sterically hindered, polar amino acids, bearing protected side chains gave insufficient results as shown in Table 2.

Table 2. Summary of experiments using BTC NMP with polar amino acids. a. After lh. at 75°C; b. After 3 coupling cycles at 65°C; c. After 2 coupling cycles at 65°C; d. Analyzed according to the general procedure D for hydrophobic peptides.

The total failure of these amino acids and a large variety of other polar and aromatic Fmoc AA's to couple to the more hindered BU's-peptidyl-resin, and also the fact that no reaction was obtained with Fmoc-Asn(Trt) and Fmoc-His(Trt) even with Ala BU-peptidyl-resin in NMP, required changing to a solvent inert to the reaction. Indeed, double coupling of Fmoc-Val and Fmoc-Ile to N^Cco-carballyloxypropyl) Ala (AlaC3) and Na(o-carballyloxypropyl) Leu (LeuC3) -peptidyl-resins using BTC in THF for only lh. at 50®C afforded the desired peptide in 100% conversion and with no detectable racemization. These results prompted us to undertake a wide synthetic effort which includes the coupling of all proteinogenic Fmoc-AA's (except Gly) to a large variety of BU's-peptidyl-resins and also to N-Me-Ala and to N-Me-Phe-peptidyl-resins, where the size and sequence of the peptidyl moiety varies. The results are shown in Table 3 and summarized in Table 4.

Table 3. Summary of difficult coupling using BTC/THF, Dioxane, Diglyme or DCP 42 Phe AlaN2-Rink 100 678.49 679.9 20.45 x 43 Phe N-MePhe-N- 100 528.2 529.9 22.06 Y MePhe-Rink 44 PheCl N-MeAla-Ala- 100 616.8 617.8 16.25 x AlaN2-Rink 45 PheC2 N-MeAla-Ala- 100 502.9 503.9 9.45 x AlaN3-Rink 46 PheC2 N-MeAla-Ala- 100 488.2 489.2 9.79 x AlaN2-Rink 47 N-MePhe N-MePhe-Rink 100 528.2 529.9 22.06 Y 48 N-MePhe N-MePhe-N- 100 500.18 501.2 18.52 x MePhe-Rink 49 Pro ValC3-Thr-Rink 100 440.31 441.2 25.95 Y 50 Ser(tBu) LysC3-Thr-Rink 100 459.32 460.1 15.61 Y 51 Thr(tBu) AlaC2-Peptide 100 1587.43 1588.7 19.25 Y -Rink 52 Thr(tBu) AlaC3 -Peptide 100 1601.46 1602.7 19.67 Y -Rink 53 Thr(tBu) ValC3-Thr-Rink 53 444.3 445.1 24.35 Y 54 Trp(Boc) ValN3-Trp-Rink 100 629.38 630.2 20.01 x 55 Τφ (Boc) ValC3-Thr-Rink 100 529.33 530.1 17.80 x 56 Tyr(tBu) LeuN3 - Amb- Ala- 100 1322.6 1323.6 19.05 x Arg-Rink 57 D-Tyr(tBu) AlaN3-Rink 100 1716.0 1716.8 14.76 x 58 Val AlaC3-Thr-Rink 100 414.29 416.0 11.93 x 59 Val LysC3-Thr-Rink 92 471.35 472.1 19.49 Y 60 Val ValC3-Thr-Rink 66 442.32 443.1 14.94 x 61 PheC3 Thr-Rink 100 391.23 392.9 10.64 x Product mass data: In entries 17, 18, 19, 20, 22, 31, 37, 44, 45, 46, 57 data correspond to the cyclic peptides. In entries 42, 51, 52, 56 data corresponds to the Allyl/Alloc protected peptides.

In entries 22, 41, 48, 51, 52 data correspond to the linear peptides.

In entries 43, 47 data correspond to the Ac-N tripeptide.

Peptide sequences: 17A PheC2-N e Ala- Ala- AlaN3 -NH2, 17B. GlyCl-Ala-Lys-(D)Ala-Ala-AlaN3- NH2; 18. Ala-Ala-Lys-(D)Ala-Ala-AlaC2- NH2; 19A (D)Ala-AlaN2-Ala-Lys-(D)Ala-Ala-AlaC3- H2 19B. AlaN2-Ala-Lys-(D)Ala-Ala-AlaC3-NH2; 20A. GlyCl-Ala-Lys-(D)Ala-Ala-AlaN2-NH2, 20B. PheC2-N-MeAla-Ala-AlaN2-lSIH2; 22 (D)Ala-AlaN2-Ala-Lys-(D)Ala-Ala-AlaC3- NH2; 11. Τ 03-8βΓ-Ο1υ-Τ^-ΐ6υ-Ρ1ιβΝ2-Ο1η- Η2; 37. PheCl-Phe-Phe-(D^-(L)Lys-PheN2-MI2 38. PheCl-Phe-Phe-(D^-(p)Lys-PheN2-NH2 39. PheCl-Phe-Leu-(D)T -(D)Lys-PheN2-NH2; 41. Biotin-Tφ-Arg-Lys-(D)AΓg-Phe-AlaC3-Leu-Arg-(D)Tyr-AlaN3-lSIH2; 42. PheCl-Ala-Phe-AlaN2-NH2; 47. Phe-NMePhe-NMePhe-NH2; 48. NMePhe-lSIMePhe- MePhe- H2; 51. Thr-AlaC2-Ser-Glu-Asn-His-Leu-Arg-His-Ala-LeuN3-Ser-NH2; 52. Thr-AlaC3-Ser-Glu-Asn-His-Leu-Arg-His-Ala-LeuN3-Ser- H2; 56. TφC3-SeΓ-Glu-Tyr-LeuN3-Amb-Ala-Arg- NH2. 57. Biotin-Tφ-Arg-Lys-(D)Arg-Phe-AlaC3-Leu-Arg-(D)Tyr-AlaN3-NH2; HPLC analysis methods: x. Analyzed according to the general procedure D for hydrophobic peptides. Y. Analyzed according to the general procedure D for hydrophilic peptides.

Table 4: Summary of Difficult Coupling Reactions Using the BTC Meth Footnotes to table 4: 1. Coupling was performed twice for 1 hour at 50°C, with 5 equivalents AA (0.14 M), 1.5 equivalents BTC (0.33 equivalents per AA), and 14 equivalents Collidine in solvent inert to this reaction. 2. Numbers indicate the position (from the C-terminal) of the BU to which the AA was coupled with 80% conversion (unless specified different), based on HPLC and mass spectra analysis.

Notes: a. Only 5-10% conversion, b. No reaction, c. Only 20% conversion with epimerization, d. Six coupling cycles, e. Only 50% conversion.

As can be seen in Table 3 the conversion of most of the couplings were quantitative regardless of the incoming Fmoc-AAs, the structure of the BU, the sequence and the size of the peptidyl moiety. In the following description the bold numbers in brackets denote the example numbers, as per table 3.

Three peptides (26, 36 and 53) gave below 70% conversion. It should be noticed that these couplings were not optimized.

Contrary to the pre-formed acid chloride method, where there was a major problem with the lability of the side chain protection groups, using the methods of the current invention the coupling of the following Fmoc-AAs gave substantially complete conversion: Arg(Pmc) (23), Asp(t-Bu) (24), Cys(Trt) (25), Lys(Boc) (34, 35), Ser(t-Bu) (50), Thr(t-Bu) (51, 52), Trp(Boc) (54) and Tyr(t-Bu) (56, 57). Since the side-chain Boc protecting group is easily removed by acids, we have monitored its stability by the Keiser test during the BTC mediated coupling of Fmoc-Lys(Boc) (34, 39). During all these couplings the Keiser test was negative and the desired backbone cyclic peptides were obtained in excellent yields without side products in the crude. From these results it can be concluded that coupling with BTC under the conditions described above does not remove sensitive side chain protecting groups normally used in solid phase peptide synthesis with Fmoc chemistry.

We have shown that most of the Fmoc-AA chlorides do not racemize during couplings to BU-peptidyl-resin in various solvents with or without collidine as base. The assessment of the degree of racemization during couplings mediated by BTC in solvent inert to this reaction is based on the following results: (a) as shown in Table 1 when racemization occurs, two peaks with identical mass were found by HPLC. In all the peptides shown in Table 3, including those peptides that gave low yields ( 21, 23, 26, 28, 33, 36, 53, 58, 59) only the major peak gave the desired mass, (b) coupling of Fmoc-Lys(Boc) and Fmoc-D-Lys(Boc) to PheN2 BU-resin , further assembly of the peptides, Allyl/ Alloc deprotection, cyclization and removal from the resin yielded two diastereomeric backbone cyclic peptides (37, 38) in quantitative conversion. The HPLC profile of each individual crude diastereomer showed a single distinct peak with the same mass and different retention times from one another. Moreover, coinjection to Capillary Electrophoresis of the two diasteromers 37 & 38 gave two separate peaks. The lack of racemization during the coupling of Fmoc-AAs chlorides to BU-peptidyl-resin is due to the high reactivity of acid chlorides to the nucleophilic acyl substitution compared to the slower oxazolon formation. Since the BTC mediated coupling in solvents inert to this reaction proceeds via in-situ acid chloride formation, it is not surprising that generally this coupling proceeds without racemization..

In order to find the limitations of BTC to promote difficult couplings we synthesized peptides 44-46 in which various Phe BUs were coupled to N-Me-Ala-peptidyl-resin. In addition, in peptide 43 Fmoc-Phe was coupled to N-Me-Phe-N-Me-Phe-resin and in peptide 48 Fmoc-N-Me-Phe was coupled to N-Me-Phe-N-Me-Phe-resin. All these repetitive couplings proceeded in short time with quantitative conversion and leads to the conclusion that BTC is the reagent of choice to promote difficult couplings in SPPS.

In most of the peptides presented in Table 3, the coupling was performed on di-peptidyl BU-resin. In order to test the capability of BTC to effect couplings to BU attached in other positions along the peptide chain, coupling was performed in positions 4-6 and 11 (peptides 56, 41, 22, 51 and 52, respectively). Table 4 depicts an overview of the peptides synthesized by BTC coupling and correlates the type of the incoming BU with the position of coupling along the peptide chain. While couplings to positions 4-6 proceeded under the normal conditions, coupling to position 11 needed six cycles to achieve quantitative conversion.

The use of BTC for the couplings of Fmoc-His(Trt)and FmocAsn(Trt) failed. Where the coupling of Fmoc-His(Trt) gave only 20% conversion with total racemization, there was no coupling with Fmoc-Asn(Trt).

Reference List Andre, F., Marraud, M., Tsouloufis, T., Tzartos, S. J. and Boussard, J. (1997) J Pep. Sci. 3, 429-441.

Bitan, G, Muller, D., Kasher, R, Gluhov, E. V. and Gilon, C. (1997a). J. Chem. Soc., Perkin trans. 7 1501.

Bitan, G, Sukhotinsky, I., Mashriki, Y., Hanani, M., Selinger, Z. and Gilon, C. (1997b). J. Pept Res. 49 421.

Byk, G, Halle, D., Zeltser, I., Bitan, G, Selinger, Z. and Gilon, C. (1996). J. Med. Chem. 39 3174.

Carpino, L. A., El-Faham, A., Minor, C. and Albericio, F. J. (1994). J. Chem. Soc., Chem. Commun. 301-203.

Carpino, L. A, Beyerman, M., Wenschuh, H. and Bienert, M. (1996) Acc. Chem. Res. 29, 268-274.

Coste, I, Dufour, M.-N., Pantaloni, A. and Castro, B. (1990). Tetrahetron Lett. 31 669. Cotarca, L., Delogu, P., Nardelli, A. and Sunjic, V. (1996). Synthesis 553-576.

Councler, C. (1880). Ber. Dtsch. Chem. Ges. 13 1697.

Fridgen, L. N. and Prol, J. J. (1989). J Org. Chem. 54 3231.

Gilon, C, Halle, D., Chorev, M., Selinger, Z. and Byk, G. (1991). Biopolymers 31 745-750.

Gilon, C, Huonges, M., Matha, B., Gellerman, G, Hornik, V., Rosenfeld, R., Afargan, M., Amitay, O., Ziv, O., Feller, E., Gamliel, A., Shohat, D., Wanger, M., Arad, O. and Kessler, H. (1998). J. Med. Chem. 41 919-929.

Henklein, P., Beyermann, M., Bienert, M. and Knorr, R. (1990). In ""Peptides 1990" Proc. of the 21th European Peptide Symposium" (E. Giralt and D. Andreu, eds), pp. 67. ESCOM Leiden.

Kaljust, K. and Unden, A. (1994). Int. J. Pep. Prot. Res. 43 505-511.

Mukaiyama, T. (1979). Angew. Chem., Int. Ed. Ingl. 18 707-808.

Muller, D., Zeltser, I., Bitan, G. and Gilon, C. (1997). J. Org. Chem. 62 411-416.

Spencer, I, Antonenko, V., Delaet, N. and Goodman, M. (1992). Int. J. Pep. Prot. Res. 40 282-293.

Abbreviations AA, amino acid ACN, acetonitrile AIB, Aminisobutyric acid Alloc, Allyloxycarbonyl Amb, 3 Amino methyl benzoic acid Arg, Arginine Asp, Aspartic acid Boc, t-butoxycarbonyl BOP, benzotriazole-l-yl-oxy-tris-(dimethylamino) iphosphonium hexaflourophosphate BTC, bis-(trichloromethyl)carbonate BU, building unit Cys, Cysteine DCM, dichloromethane DTEA, diisopropylethylamine DOT A, tetraazacyclodecanetetraacetic acid DTP A, diethylentriaminepenta acetic acid Fmoc, fluorenylmethoxycarbonyl Gin, Glutamine Glu, Glutamic acid HATU, O-(7-azabenzotriazol-l-yl)-l, 1,3,3-tetramethyluronium hexafluorophosphate HO At, l-hydroxy-7-azabenzotriazole HPPA, parahydroxy phenyl propionic acid Lys, Lysine MBHA, methylbenzhydrilamine MPPS, multiple parallel peptide synthesis N-BBC, N-backbone cyclic NMP, N-methyl pyrollidone Pmc 2,2,5,7,8-Pentamethyl chroman-6-sulphonyl Pro, Proline PyBrOP, bromo-tris-pyrrolidino-phosphonium hexafluorophosphate Ser, Serine SPPS, solid phase peptide synthesis t-Bu, t-butyl TFA, trifluoroacetic acid THF, tetrahydrofuran Thr, Threonine TIS, triisopropylsilane TOPPipU, 2-(2-Oxo-l(2H)-pyridyl)-l, 1,3,3-bispenta-methyleneuronium tetrafluoroborate Trp, Tryptophan Trt, trityl Tyr, Tyrosine UNCA, urethane- protected N-carboxyanhydrides Val, Valine ABSTRACT A process is disclosed for using triphosgene as an efficient and effective coupling reagent during peptide synthesis, by in situ generation of amino acid chloride from a protected amino acid. This process is particularly useful for the coupling to sterically hindered amino acid residues, or for other difficult couplings. Furthermore, the same reagent can be used for the derivatization of peptides by formation of an amide bond between a free amine on a peptide and a carboxylic acid, or for the coupling of an amino acid to a solid support. 26

Claims (14)

1. A process of coupling an amino acid residue to a peptide chain which comprises: (i) providing an amino acid residue having a free carboxylic group and blocked amino group, optionally having additional blocked finctional side chains; (ii) reacting the blocked amino acid with bis-(trichloromethyl)carbonate in a solvent inert to this reaction to obtain an amino acid chloride; (iii) neutralizing the free acid with an organic base; (iv) adding the resulting suspension containing the amino acid chloride to a compound selected from the group consisting of a peptide having a blocked carboxyl terminus and a free amino terminus, and a peptidyl resin having at least one free amino terminus; and (v) providing reaction conditions enabling the coupling of the amino acid chloride to the peptide to yield a peptide elongated by one amino acid residue.

2. The process of claim 1 wherein the peptide chain comprises a sterically hindered residue in the N terminal position.

3. The process of claim 2 further comprising heating the reaction mixture during the coupling of the amino acid chloride to the peptide.

4. The process of claim 1 further comprising adding a catalyst to the reaction mixture of the amino acid chloride and the peptide.

5. The process of claim 2 wherein the sterically hindered residue in the N terminal position of the peptide chain comprises a sterically hindered secondary amine.

6. The process of claim 2 wherein the sterically hindered residue in the N terminal position of the peptide chain comprises an N-alpha (co-functionalized) alkyl amino acid residue. 23 125314/2

7. The process of claim 1 wherein the inert solvent is selected form the group consisting of dioxane, tetrahydrofuran, diglyme and 1,3 dichloropropane.

8. The process of claim 1 wherein the peptide coupling further comprises multiple parallel peptide synthesis.

9. The process of claim 1 wherein the coupling agent is provided at a stoichiometric ratio of at least about one third molar equivalent of the amino acid residue.

10. The process of claim 1 wherein the base is selected from the group consisting of collidine, diisopropylethylamine, pyridine, dimethyl pyridine and quinaldine.

11. 1 1. The process of claim 1 wherein the amino group of the amino acid is blocked by a blocking group selected from the group consisting of fluorenylmethoxycarbonyl, and tert butoxycarbonyl.

12. A process of coupling an amino acid residue to a solid support which comprises: (i) providing an amino acid residue having a free carboxylic group and blocked amino group, optionally having additional blocked functional side chains; (ii) reacting the blocked amino acid with bis-(trichloromethyl)carbonate in a solvent inert to the reaction to obtain an amino acid chloride; (iii) neutralizing the free acid by addition of an organic base; (iv) adding the resulting suspension containing the amino acid chloride to a compound selected from the group consisting of a resin having at least one free amino terminus and a solid support having a functional group capable of binding the chloride; and (v) providing reaction conditions enabling the coupling of the amino acid chloride to the solid support.

13. In the in situ synthesis of peptides, the improvement which comprises synthesizing an amino acid chloride using one of bis-(trichloromethyl) carbonate, diphosgene or phosgene. 24

14. The invention of claim 13 wherein the peptides are synthesized during solid phase peptide synthesis. For the Applicants, Webb & Assoc ates Patent Attorneys 25